Purification and characterization of adrenocortical adenosine 3',5'-monoposphate-dependent protein kinases.

Abstract

In this manuscript we describe in detail the purification and biochemical and immunological characterization of cAMP-dependent protein kinases in bovine adrenal cortex, rat adrenal gland, and isolated fasciculata cells of the rat. DEAE-cellulose chromatography of bovine adrenal cortex extract yielded two major (type I and type II) cAMP-dependent protein kinase peaks and one minor cAMP-binding peak. The minor peak (peak A) eluted at 30-80 mM NaCl and corresponded to the typical type I tetrameric structure of the holoenzyme. Peak B, eluting at 80-130 mM NaCl, comprised 10-15% of the total cAMP-binding activity and was identified as dimeric type I cAMP-binding regulatory subunit of the enzyme. Peak C (major peak) eluting at high salt (130-220 mM NaCl), was different from the typical type II holoenzyme; its mol wt was relatively low (123,000), and its cAMP-binding subunit was type I rather than type II. The native enzyme contained dimeric cAMP-binding regulatory subunit and suggested the presence of only a single catalytic subunit. Based on these results and on the reduced activation of its kinase activity by cAMP, we suggest a type I trimeric structure, R I2 C, of this enzyme. Most of the bovine adrenocortical extracts (62 of 68) did not contain type II cAMP-binding regulatory subunit of the enzyme. When present, its concentration (free or part of the holoenzyme) was less than 15% of the total cAMP-dependent protein kinases. These results were further supported by the studies with rat adrenal glands and isolated fasciculata cells derived from these glands, where only the type I cAMP receptor was found. We, therefore, conclude that in contrast to the current notion, adrenal cortex contains little, if any, enzyme containing type II cAMP-binding receptor. The predominant form of the holoenzyme contains a typical type I cAMP-binding receptor, but possesses an anomalous type II-like high salt elution pattern. We suggest that the trimeric structure of this enzyme contains a typical dimeric type I cAMP-binding subunit and a single catalytic subunit, R I2 C.